The events that govern the regulation of RNA Pol II transcription in mammalian cells have remained ill-defined. The importance of these mechanisms to normal development, differentiation and cellular maintenance, as well as to various human diseases, makes this one of the preeminant problems in modern biology. The goal of this proposal is to define the molecular mechanism involved in transcriptional regulation of target genes by the steroid glucocorticoid hormones. The specific aims of this work are three-fold. First is a detailed in vivo analysis of the interaction of the cis-elements involved in regulation. Transient and stable transfection assays of test plasmids containing the appropriate cis-elements will be critical for these studies. Second is an in vitro analysis of the interaction of trans-acting nuclear factors involved in regulation. These interactions will be studied using as templates both naked DNA and chromatin. Reconstitution of regulatory events is an important part of this specific aim. Finally is an analysis of the glucocorticoid response in embryonal carcinoma cells. Both cis- and trans-elements will be studied in the context of the differentiating system provided by these cells.